CN110358745B

CN110358745B - 4-xylitol dehydrogenase mutant and application thereof

Info

Publication number: CN110358745B
Application number: CN201910777021.7A
Authority: CN
Inventors: 孙科; 沙凤
Original assignee: Suzhou Koning Polyol Co ltd
Current assignee: Suzhou Koning Polyol Co ltd
Priority date: 2019-08-22
Filing date: 2019-08-22
Publication date: 2022-04-19
Anticipated expiration: 2039-08-22
Also published as: CN110358745A

Abstract

The invention discloses a 4-xylitol dehydrogenase mutant and application thereof. The amino acid sequence of the 4-xylitol dehydrogenase mutant is SEQ ID NO: 1, wherein the amino acid sequence is obtained by mutating leucine at position 41 into methionine and isoleucine at position 179 into phenylalanine. The nucleotide sequence of the 4-xylitol dehydrogenase mutant gene is shown as SEQ ID NO: 4, respectively. The gene is introduced into escherichia coli to obtain a genetic engineering bacterium containing the gene, and the preparation of the recombinant 4-xylitol dehydrogenase is realized.

Description

4-xylitol dehydrogenase mutant and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a 4-xylitol dehydrogenase mutant and application thereof.

Background

According to the definition of rare sugars by the International sugar society (ISRS), rare sugars are "monosaccharides and their derivatives present in nature but in very small amounts". Although the rare sugar has a small content in nature, the rare sugar plays an important role in the fields of diet, health care, medicine and the like due to potential biological activity and low toxicity. With the improvement of life quality, people have higher requirements on diet and health, and pay more attention to food safety and low drug toxicity. L-xylulose (L-xylulose) is a ketopentose present in a variety of metabolic pathways in organisms, and is a rare sugar that is present in very low concentrations in nature. The L-xylulose can inhibit the activity of intestinal sucrase and maltase, can effectively inhibit the increase of postprandial blood sugar, is an ideal active ingredient of blood sugar-reducing food, and can obviously inhibit malignant tumor cells when being used for treating tumors. In addition, L-xylulose can be used as a precursor for the production of other rare sugars. For example, L-xylulose can be converted by L-rhamnose isomerase to produce L-xylose and L-lyxose.

According to the Izumoring theory, the L-xylulose can be obtained through various ways. For example, L-xylose and L-lyxose are converted into L-xylulose by isomerization with aldose isomerase; the L-ribulose acts on a hydroxyl at the C-3 position through D-tagatose 3-epimerase, and is converted into L-xylulose through epimerization; l-arabinitol and xylitol are converted into L-xylulose by oxidation of an oxidoreductase. Wherein the content of L-xylose, L-lyxose, L-ribulose and L-arabitol in nature is very little, the separation and purification are difficult, the price is extremely expensive, and the production cost of the L-xylulose is undoubtedly increased. Xylitol, as one of the most widely used rare sugars in the commercial field at present, has already been industrially produced, is relatively cheap and is readily available. The xylitol is used as a substrate, so that the production cost of the L-xylulose is greatly reduced.

The L-xylulose is produced by biotransformation, and the substrate xylitol is catalyzed and oxidized mainly by the resting cells of thalli. Usually, the cells of the cultured cells are collected and washed by filtration or centrifugation, and then suspended in a reaction system containing a substrate (mainly xylitol) and subjected to conversion production under conditions of a certain temperature, pH and shaking. The method has mild reaction conditions, is environment-friendly, has low cost and high product purity, is easy to realize large-scale production, and is the most potential biological conversion method based on long-term consideration and the main research direction of the L-xylulose production technology. The study of L-xylulose production by microbial transformation was first described in 1985, Doten et al, Connell university, USA, isolated 1 spontaneous mutant strain (Doten et al, 1985) with xylitol as the sole carbon source in the course of studying the pentitol metabolism of the bacterial plant pathogen Erwinia uredovora (now more known as Pantoea ananatis), which was detected to have the activities of 4-Xylitol Dehydrogenase (XDH) and L-xylulokinase, this novel 4-xylitol dehydrogenase, with xylitol as the substrate K_MReaching 48Mm, can catalyze the reduction reaction of L-xylulose with NADH as coenzyme. Subsequently, they screened a mutant strain of xylitol negative mutant by transposon mutagenesis, which can synthesize 4-xylitol dehydrogenase and lack L-xylulokinase activity. The mutant can convert xylitol into L-xylulose without further phosphorylation of L-xylulose into the pentose phosphate pathway (Doten et al, 1985). Finland Helsinki Dada in 2006Aarnikunnas et al succeeded in isolating and expressing the 4-xylitol dehydrogenase gene in Pantoea ananatis in E.coli (E.coli), which was the first successful cloning and expression of a bacterially-derived 4-xylitol dehydrogenase gene. It was shown by studies on the influence of 4-xylitol dehydrogenase that XDH is regulated by magnesium ions, which is most suitable for p H in the basic range (Aarnikunnas et al, 2006). The response surface method is used for optimizing the influence factor of converting xylitol into L-xylulose by resting cells of recombinant Escherichia coli, and the productivity of the L-xylulose reaches 1.09g/(gh) when 250g/L of xylitol is used as a substrate in a bioreactor (Usvalamet al, 2009).

In 1991, the group IZumori, a rare sugar research center of Japan, university of Xiangchuan, isolated and screened from soil using D-sorbitol as a sole carbon source to obtain 1 strain of Alcaligenes (Alcaligenes) capable of oxidizing talose to psicose, which has an ability to convert xylitol to produce L-xylulose in vivo (Khan et al, 1991). In 2007, the same group of rare sugar research center of university of Kangawa, Japan separated 1 strain of facultative thermophile from soil, and the resting cells of the strain were used to transform xylitol into L-xylulose, which was then identified as Bacillus pallidus (Poonerm et al, 2007). The activity of resting cells of the strain is highest in a glycine-NaOH buffer solution (pH 10) at 40 ℃, and the conversion rate reaches 85 percent when the resting cells act on 2 percent xylitol. In 2010, Takata et al successfully expressed the 4-xylitol dehydrogenase gene in Bacillus bigbiensis in vivo in E.coli. The recombinant escherichia coli resting cells are placed in a glycine-sodium hydroxide buffer solution with the pH value of 11.0, 5% of xylitol is used as a substrate to produce L-xylulose, and the conversion rate of the xylitol reaches 35% after 24 hours (Takata et al, 2010).

So far, the domestic research on the production of L-xylulose is very little, and only two documents are reported. Zhangyubao et al isolated and screened 1 strain ZN-14 which can oxidize xylitol and convert the strain to produce L-xylulose from soil in 2014, and identified the strain as Bacillus megaterium (Zhangyubao et al, 2014). Resting cells of the strain are converted for 24 hours at 37 ℃ by using a xylitol solution (20g/L, pH 9.0) as a substrate, the conversion rate reaches 26.62%, and the enzyme activity is 2.6U/m L. In 2019, 4-xylitol dehydrogenase gene derived from Pantoea ananatis is expressed and fermented in recombinant bacillus subtilis by Zhu Wen Hui et al and reaction conditions are optimized (Zhu Wen Hui et al, 2019), and the optimal reaction conditions are obtained through resting cell reaction, wherein the mass concentration of a substrate is 20g/L, a buffer solution is a glycine-NaOH solution (pH is 10.0), and the reaction temperature is 45 ℃. The final enzyme activity obtained under the conditions is 5.183U/L, which is 231.2% higher than that of LB culture medium, and the conversion rate reaches 17.74%.

So far, only 4-xylitol dehydrogenase of four different microbial sources of wild type (Pantoea ananatis, Alcaligenes latus, Bacillus xanthus and Bacillus megaterium) mentioned above has been reported, and these XDH have generally poor thermal stability and low activity, which results in low yield of L-xylulose, limiting the industrial application range thereof. Generally, in the industrial production of rare sugars, higher operating temperatures are required. This is because higher reaction temperatures can bring a number of advantages: improved reaction efficiency (higher reaction rate and lower diffusion limitation), reduced solution viscosity, increased reaction stability, higher yields (increased solubility of substrate and product, driving the equilibrium of the reaction towards endothermic reactions), and reduced microbial contamination, among other things. The method is characterized in that a protein directed evolution technology is utilized to transform wild type 4-xylitol dehydrogenase so as to obtain a 4-xylitol dehydrogenase mutant which has high activity and good thermal stability and is suitable for industrial application, and the method is the key of the L-xylulose preparation industry by a biological method.

Reference to the literature

Doten R.C.,Mortlock R.P.Characterization of xylitol-utilizing mutants of erwinia uredovora.Bacteriology,1985,161(2):529-533

Doten R.C.,Mortlock R.P..Production of D-and L-xylulose by mutants of Klebsiella pneumoniae and Erwina uredovoru.Appl.Environ.Microbial.,1985,(49):158-162

Doten R.C.,Mortlock R.P..Inducible xylitol dehydrogenases in enteric bacteria.Bacteriol.1985,162(2):845

Aarnikunnas J.S.,Pihlajaniemi A.,Palva A.,Leisola M.,

A..Cloning and expression of a xylitol 4-dehydrogenase gene from Pantoea ananatis.Appl Environ Microbiol,2006,(72):368–377

Usvalampi A.,Kiviharju K.,Leisola M.,

M..Factors affecting the production of L-xylulose by resting cells of recombinant Escherichia coli.Ind.Microbiol.Biotechnol.2009,(36):1323-1330

Khan A.R.,Tokunaga H.,Yoshida K.,Izumori K..Conversion of xylitol to L-xylulose by Alcaligenes sp.701B-Cells.Fermention Bioengineering,1991,72(6):488-490

Poonperm W.,Takata G.,Morimoto K.,Granstrom T.G.,Izumori K..Production of L-xylulose from xylitol by a newly isolated strain of Bacillus pallidus Y25and characterization of its relevant enzyme xylitol dehydrogenase.Enzyme and Microbial Technology.2007,(40):1206-1212

Takata G，Poonperm W，Morimoto K，et al.Cloning and overexpression of the xylitol dehydrogenase gene from Bacillus pallidus and its application to L-xylulose production.Journal of the Agricultural Chemical Society of Japan.2010,74(9):1 807-1 813.

Zhangyubao, Zhao Xiang Ying, Yangli Limnu, etc. one strain of L-xylulose producing strain is separated, screened and identified, food science, 2014, 35(1) 199 and 203.

Fermentation and reaction condition optimization of recombinant bacillus subtilis expressing 4-xylitol dehydrogenase from Zhu Wen Hui, Meng, Jianbo, Zuo, food and fermentation industries, 2019,45(9):21-28

Disclosure of Invention

The present inventors have conducted previous studies and have found that 4-xylitol dehydrogenase derived from Klebsiella oxytoca (Klebsiella oxytoca), although having high activity, does not obtain good catalytic effect due to low thermostability. Therefore, it is required to develop a mutant of 4-xylitol dehydrogenase (GenBank: STR65190.1) having improved thermostability by using site-directed mutagenesis to improve its efficiency in the catalytic production of L-xylulose.

In order to realize the purpose, the invention utilizes molecular docking to establish a compound model of a substrate (xylitol) and an enzyme (4-xylitol dehydrogenase), analyzes the structural mechanism of the combination of the substrate and the enzyme, selects key residues which can influence the thermal stability of the 4-xylitol dehydrogenase on xylitol, and obtains a 4-xylitol dehydrogenase mutant by site-directed mutagenesis. The amino acid sequence of the 4-xylitol dehydrogenase mutant is SEQ ID NO: 1, wherein the amino acid sequence is obtained by mutating leucine at position 41 into methionine and isoleucine at position 179 into phenylalanine.

Another object of the present invention is to provide a method for preparing the mutant of 4-xylitol dehydrogenase.

It is still another object of the present invention to provide uses of the 4-xylitol dehydrogenase mutant.

The purpose of the invention can be realized by the following technical scheme:

the 4-xylitol dehydrogenase mutant gene, the wild type gene of which is from Klebsiella (GenBank: STR65190.1), is obtained by site-directed mutagenesis, and the nucleotide sequence of the gene is shown as SEQ ID NO: 4, respectively.

A4-xylitol dehydrogenase mutant has an amino acid sequence shown as SEQ ID NO: 2, respectively.

A recombinant vector comprising the 4-xylitol dehydrogenase mutant gene. The recombinant plasmid is constructed by connecting the nucleotide sequence of the 4-xylitol dehydrogenase gene of the invention to various vectors by a method conventional in the art, and the recombinant plasmid is selected from the group consisting of pET-22b (+), pET-3a (+), pET-3d (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pQE2, pQE9, pQE30, pQE 31, pR A, pRSET-B, pRSET-C3552-C (+), and, pGEX-5X-l, pGEX-6p-2, pBV220, pTrc99A, pTwin1, pEZZ18, pKK232-18, pBR322, pUC-18 or pUC-19. More preferably, the above recombinant plasmid is pET-28a (+). Meanwhile, for expression in Bacillus subtilis, preferably, a recombinant plasmid selected from the group consisting of pWB980, pHT43, pBE2, pMUTIN4, pUB110, pE194, pMA5, pMK3, pMK4, pHT304, pHY300PLK, pBest502, pDG1363, pSG1154, pAX01, pSAS144, pDL, pDG148-stu, pDG641, pUCX05-bgaB, pHT01, pUB110, pTZ4, pC194,. phi.1 and. phi.105 can be used. More preferably, the above recombinant plasmid is pMA 5.

A genetic engineering bacterium for producing the 4-xylitol dehydrogenase mutant comprises the 4-xylitol dehydrogenase mutant gene or the recombinant vector.

The host cell of the genetic engineering bacteria comprises prokaryotic cells, yeast or eukaryotic cells; preferably, the prokaryotic cell is an escherichia coli (e.coli) cell or bacillus subtilis. More preferably, the host cell is an E.coli BL21(DE3) cell.

The invention relates to a 4-xylitol dehydrogenase mutant gene, a recombinant vector and application of a genetic engineering bacterium in preparing L-xylulose.

A preparation method of a 4-xylitol dehydrogenase mutant comprises the following steps: culturing the genetic engineering bacteria to obtain the recombinant expression 4-xylitol dehydrogenase mutant.

The 4-xylitol dehydrogenase mutant provided by the invention is used for preparing L-xylulose by converting xylitol.

Catalytic reaction conditions, substrate concentration of 30g/l, reaction time of 6 hours and temperature of 45 ℃.

Advantageous effects

Compared with the 4-xylitol dehydrogenase in the prior art, the 4-Xylitol Dehydrogenase (XDH) mutant of the invention has better thermal stability under the condition of keeping the original high catalytic activity, and the two points are good performance for catalyzing xylitol to produce L-xylulose under the condition of higher temperature, so that the consumption of the catalyst can be greatly reduced and the reaction time can be shortened in industrial production, thereby reducing the production cost. According to the examples of the present invention, 80% of the enzyme activity of mutant XDH was retained after 2 hours of incubation at 45 ℃. In an experiment for producing L-xylulose by using recombinant genetic engineering bacteria to convert xylitol sugar, when the substrate concentration is 30g/L, the conversion rate of the XDH mutant is 88.7 percent, which is improved by nearly 2 times compared with the XDH mutant, and the enzyme activity is high, the heat stability is good, and the catalytic efficiency is high.

The L-xylulose thus produced can be effectively used for research of foods or medicines.

Drawings

FIG. 1 is a graph showing a NADH standard curve under the conditions of the embodiment of the present invention.

FIG. 2 is a graph showing the effect of temperature on XDH and mutant activity thereof under the conditions of an embodiment of the present invention.

FIG. 3 is a graph showing the thermostability of XDH and its mutants under the conditions of an embodiment of the present invention.

Detailed Description

Hereinafter, the present invention will be described in more detail with reference to specific examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: establishment of genetically engineered bacteria

The 4-xylitol dehydrogenase gene fragment was commercially synthesized based on the 4-xylitol dehydrogenase gene XDH (GenBank: STR65190.1) included in NCBI, and the nucleotide sequence of the fragment (the NdeI and BamHI restriction enzyme fragments on both sides of the fragment) was extended by PCR using the gene fragment as a template as shown in SEQ ID No. 3. And the gene was inserted into pET-28a plasmid using NdeI and BamHI restriction enzyme sites, thereby producing recombinant plasmid pET-28 a-XDH. The recombinant plasmid is transformed into Escherichia coli BL21(DE3) by a conventional transformation method to establish the 4-xylitol dehydrogenase gene engineering bacterium. The recombinant strain E.coli BL21(DE3)/pET-28a-XDH containing the wild type XDH gene obtained by transformation was stored in an ultra-low temperature refrigerator at-80 ℃. Wherein the primers for PCR amplification of wild type XDH gene are as follows:

the upstream primer is 5'-CGCGGCAGCCATATGATGATTGAACCTGTGGCCTG-3' (SEQ ID NO.5)

The downstream primer is 5'-CTCGAATTCGGATCCTTACTTATCAGCTTTAATAA-3' (SEQ ID NO.6)

Example 2: site-directed mutagenesis

The primers for site-specific mutagenesis are designed by using Primer premier 5.0, and the principle of Primer design is as follows: the 5' end of the forward and reverse amplification primers comprises a 15-21bp reverse complementary region, the length of each primer non-complementary region is at least 15bp, and the mutation to be introduced is contained in the complementary region. The mutant primers are shown in Table 1.

TABLE 1 mutant primers

Site-directed mutagenesis takes pET-28a-XDH or pET-28a-XDH (L41M) recombinant plasmid as a template, PrimerStar Mix is used for full-plasmid amplification, a reaction system is set according to the table 2, the template in a PCR reaction system is removed by digesting an amplification product through Dpn I enzyme, and then 5 'end and 3' end are subjected to homologous recombination under the catalysis of recombinase to complete the cyclization of the plasmid. Finally, the circularized amplification product was transferred to E.coli BL21(DE3) host bacteria, spread on kanamycin-containing plates, and cultured overnight in a 37 ℃ incubator.

TABLE 2 site-directed mutagenesis System

The PCR procedure was: pre-denaturation at 95 ℃ for 300s, denaturation at 98 ℃ for 10s, annealing at 65 ℃ for 15s, extension at 72 ℃ for 300s, reaction for 30 cycles, extension at 72 ℃ for 5min, and final heat preservation at 4 ℃. After the PCR reaction, the PCR product was detected by 0.8% agarose gel electrophoresis. Then 1. mu.l of Dpn I was added to each PCR tube, gently mixed and then placed in a 37 ℃ metal bath for 2 hours, and then the digested amplification product was subjected to recombination reaction according to the formula in Table 3.

TABLE 3 recombination reaction System

The next day, three recombinant E.coli BL21 strains containing mutant plasmids were selected from the plates, and the above recombinant bacteria were inoculated from the plates into 50mL shake tubes containing 5mL liquid LB medium (LB (g/L): peptone 10, sodium chloride 10, yeast extract 5) containing the corresponding resistance, respectively, and the corresponding resistance was added, and the cells were incubated on a shaker at 37 ℃ for 12 hours at 200 rpm. After the culture was completed, the plasmid was extracted and sequenced by Kinshire. Finally, the sequencing result is compared with the wild type enzyme protein nucleic acid sequence to determine whether the mutation is successful.

According to the method, single site mutation is firstly carried out to obtain recombinant expression bacteria E.coli BL21(DE3)/pET-28a-XDH (L41M), then secondary mutation is carried out by taking the recombinant plasmid pET-28a-XDH (L41M) subjected to primary mutation as a template, and finally the double site mutation recombinant expression bacteria E.coli BL21(DE3)/pET-28a-XDH (L41M + I179F) containing the XDH mutant with correct sequencing is obtained.

Example 3: fermentation culture of recombinant escherichia coli

The recombinant bacteria E.coli BL21(DE3)/pET-28a-XDH and E.coli BL21(DE3)/pET-28a-XDH (L41M + I179F) obtained in example 1 and example 2 were inoculated into 5mL of LB medium containing kanamycin (50. mu.g/mL), respectively, and cultured with shaking at 37 ℃ and 200rpm for 8 hours. 1mL of the cell culture was transferred to a 250mL shake flask containing 50mL of TB fermentation medium ((g/L): peptone 15, yeast extract 25, sodium chloride 10, kanamycin-containing 50. mu.g/mL), cultured with shaking at 37 ℃ and 200rpm for 3 hours, further cultured at 25-30 ℃ and 200rpm for 12-16 hours, and then centrifuged at low temperature (8000rpm, 10min, 4 ℃) to collect the cells, washed twice with phosphate buffer (pH7.5, 100mmol/L), dispersed in 1mL of the same precooled buffer, and sonicated in ice water. Centrifuging (8000rpm, 30min,4 ℃), and discarding the thallus fragment to obtain crude enzyme solution of wild type XDH and its mutant.

Example 4: purification of XDH

For purification of the above XDH and its mutants. The AKTA prime chromatography system of GE was used, and 5mL HisTrapHP nickel column was used for affinity chromatography. The chromatographic column is pre-balanced by pH 8.5, 0.5M NaCl, 20mM imidazole and 20mM sodium phosphate buffer (buffer A), the elution buffer is pH 8.5, 0.5M NaCl, 0.5M imidazole and 20mM phosphate buffer (buffer B), gradient elution is carried out by adopting 0-100% buffer B, the total elution time is 30min, then the collected active protein is further desalted and removed of foreign protein by a Sephacryl S-300 gel column, the monitoring is carried out by a 280nm ultraviolet detector, and the target protein is collected at a high value. And (4) freeze-drying the purified enzyme to obtain freeze-dried powder for measuring the protein concentration and activity.

Example 5: enzyme Activity measurement of XDH

XDH requires NAD for the oxidation of xylitol to L-xylulose⁺As a coenzyme and NADH was generated, the enzyme activity of XDH was calculated by measuring the amount of change in absorbance at 340nm of the reaction system per unit time. A reaction system for detecting enzyme activity is arranged in an EP tube: 1.5M xylitol solution 100. mu.L, 2mM NAD⁺Solution (freshly prepared with 100mM sodium phosphate buffer, pH 8.5)100 μ L, 10mM β -mercaptoethanol 100 μ L, 500mM sodium phosphate buffer (pH 8.5)400 μ L, dd H₂O200 mu L; control was 1.5M xylitol solution 0. mu.L, 2mM NAD⁺Solution 100. mu.L, 10 mM. beta. -mercaptoethanol 100. mu.L, sodium phosphate buffer (pH 8.5) 400. mu.L, dd H₂O300. mu.L. Beta-mercaptoethanol is added in the system as a reducing agent, so that the function of preventing mutual crosslinking of disulfide bonds is realized, and the activity state of the enzyme can be maintained.

After the reaction solution was mixed well by using a pipette, the mixture was transferred to a 96-well microplate, 200. mu.l of the mixed reaction solution was added to each well, and 3 sets of 200. mu.l parallel reaction systems were provided, respectively. After the microplate reader is kept at 30 ℃ for 5 minutes, adding an equal amount of pure enzyme solution to activate the reaction, and immediately detecting the change of the absorbance value at the wavelength of 340nm as shown in the formula (1):

the specific activity of the enzyme is calculated as follows:

wherein V represents the total volume of the reaction system (200. mu.L); d-dilution factor; v-volume of enzyme solution (5. mu.L); c-protein concentration (mg/mL); ε is the molar extinction coefficient of NADH (when the optical path length is 1cm, ε is 6.220 mM)^-1cm^-1) The method is characterized in that the slope of an NADH standard curve equation (shown in figure 1) is adopted for correction, a certain amount of NADH is accurately weighed, NADH solutions with different concentrations are prepared by diluting different multiples, the absorbance value of the NADH solution at 340nm is measured, a linear equation is fitted, the abscissa is the NADH concentration, and the ordinate is the absorbance value at 340 nm. The amount of enzyme required to produce 1. mu. mol NADH per minute was taken as one activity unit (U). Protein quantification was determined using the TaKaRa BCA kit.

Experimental results show that the specific activity of the wild type XDH for catalytic conversion of xylitol is 92.6U/mg of freeze-dried powder, while the enzyme specific activity of the mutant XDH reaches 90.5U/mg of freeze-dried powder, and the high activity of the mutant XDH is only slightly reduced compared with that of the wild type XDH.

Example 6: effect of temperature on XDH mutant Activity

(1) Effect of temperature on Activity of wild-type XDH and mutants thereof

The oxidation activity of XDH purified in example 4 using xylitol as a substrate was measured at various temperatures (25 ℃ to 60 ℃) in the same manner as in example 5, and the pH of the buffer in the reaction system was 9.0. And drawing an XDH temperature-relative activity curve by taking the highest enzyme activity as 100 percent and the corresponding temperature as the optimal temperature. The results (see FIG. 2) show that the optimum temperature of wild type XDH was 45 ℃ and that the enzyme activity rapidly decreased when the reaction temperature exceeded 50 ℃, whereas the optimum temperature of XDH mutant was 50 ℃.

(2) Study of thermostability of wild-type XDH and its mutant

The wild type XDH purified in example 4 and its mutant enzyme were subjected to heat treatment in water baths at 45 ℃ and 50 ℃ respectively, and samples were taken at intervals to determine the residual enzyme activity. And drawing a temperature-relative activity curve of the XDH mutant by taking the initial enzyme activity as the highest enzyme activity 100%. The results (as shown in FIG. 3) show that the wild type XDH was maintained at 45 ℃ for 2 hours until the enzyme activity was reduced to below 60%, while the mutant was maintained at 45 ℃ for 2 hours until 80% of the enzyme activity was retained.

Example 7: production of L-xylulose by converting xylitol sugar with recombinant gene engineering bacterium enzyme

(1) Two groups of resting cell transformation experiments: to 1mL of the reaction system, 1mL of 30g/L xylitol dissolved in glycine-NAOH buffer (50mM, pH 9.0) was added, and 50mL of recombinant E.coli BL21(DE3)/pET-28a-XDH fermentation broth obtained by fermentation culture as described in example 3 was centrifuged to collect the cells, incubated at 45 ℃ for 6 hours, and then boiled for 10 minutes to terminate the enzymatic reaction.

1mL of xylitol dissolved in glycine-NAOH buffer (50mM, pH 9.0) at a concentration of 30g/L was added to 1mL of the reaction system, and 10mL of the recombinant bacterium E.coli BL21(DE3)/pET-28a-XDH (L41M + I179F) fermentation broth obtained by fermentation culture as described in example 3 was centrifuged to collect the cells, incubated at 45 ℃ for 6 hours, and then boiled for 10 minutes to terminate the enzymatic reaction.

(2) And detecting the generation amount of the L-xylulose by using a cysteine carbazole colorimetric method. After the conversion solution is properly diluted, 1mL of conversion solution is transferred, 0.2mL of cysteine hydrochloride solution with the concentration of 15g/L and 6mL of sulfuric acid solution with the volume fraction of 70% are added, 0.2mL of 1.2g/L carbazole alcohol solution is immediately added after shaking up, water bath at 70 ℃ is carried out for heat preservation for 10min, and cooling is carried out for 10 min. Zeroed with a blank and absorbance measured at 560 nm. The xylulose content produced was calculated by a standard curve.

After 6 hours of reaction, the conversion rate of L-xylulose catalytically produced by the recombinant strain containing the wild type XDH gene is 45.2%, and the conversion rate of the mutant XDH is 88.7%, which is improved by nearly 2 times compared with the conversion rate, thereby showing that the catalytic activity of the mutant XDH at higher temperature is obviously higher than that of the wild type XDH.

Sequence listing

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<120> 4-xylitol dehydrogenase mutant and use thereof

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<400> 3

atgattgaac ctgtggcctg ctgcctgcat gggctgaaat cagccaatat tcacgctaaa 60

gataccgttc tggttctggg gtccggcacg attggtctgg tgagcgctca gctcgccgct 120

ttaaaaggcg catcgaccgt catcgtatcc gatctctcgc gcttcaaacg tgaactggcg 180

ttaaaggtcg gcgtgaccca tgcgattgat cccgccaatg aaaatcttga aatgcgggtg 240

gcggaaatta cgcatgataa ggggcctgat gtggtgatca ttgcggctgg cgtatcatcg 300

ctggtttctc aggcggtgaa tattgttcgt cgcggcggca ggatcgtagt gttctctccg 360

tttgataaaa acccggtggt ggagattgac gccagccgct tgttccgcga cgaaatatct 420

atcgtcggca cctattcact gacgccttat gaaatgaaag aagctattga aatcgtagag 480

aaagataaaa ttaacaccag agatatgatt actcatacct ggccgctttc tcgtattggt 540

gaagctattg aatttgcggc aaatcctgaa aatgacgtgc ttaaagtgat tattaaagct 600

gataagtaa 609

<210> 4

<211> 609

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgattgaac ctgtggcctg ctgcctgcat gggctgaaat cagccaatat tcacgctaaa 60

gataccgttc tggttctggg gtccggcacg attggtctgg tgagcgctca gctcgccgct 120

atgaaaggcg catcgaccgt catcgtatcc gatctctcgc gcttcaaacg tgaactggcg 180

ttaaaggtcg gcgtgaccca tgcgattgat cccgccaatg aaaatcttga aatgcgggtg 240

gcggaaatta cgcatgataa ggggcctgat gtggtgatca ttgcggctgg cgtatcatcg 300

ctggtttctc aggcggtgaa tattgttcgt cgcggcggca ggatcgtagt gttctctccg 360

tttgataaaa acccggtggt ggagattgac gccagccgct tgttccgcga cgaaatatct 420

atcgtcggca cctattcact gacgccttat gaaatgaaag aagctattga aatcgtagag 480

aaagataaaa ttaacaccag agatatgatt actcatacct ggccgctttc tcgttttggt 540

gaagctattg aatttgcggc aaatcctgaa aatgacgtgc ttaaagtgat tattaaagct 600

gataagtaa 609

<210> 5

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cgcggcagcc atatgatgat tgaacctgtg gcctg 35

<210> 6

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctcgaattcg gatccttact tatcagcttt aataa 35

<210> 7

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cagctcgccg ctatgaaagg cgcatcgacc gtcat 35

<210> 8

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtcgatgcgc ctttcatagc ggcgagctga gcgct 35

<210> 9

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggccgctttc tcgtattggt gaagctattg aattt 35

<210> 10

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aatagcttca ccaatacgag aaagcggcca ggtat 35

Claims

1. A4-xylitol dehydrogenase mutant, characterized in that: the amino acid sequence of the 4-xylitol dehydrogenase mutant is SEQ ID NO: 1, wherein the amino acid sequence is obtained by mutating leucine at position 41 into methionine and isoleucine at position 179 into phenylalanine.

2. A4-xylitol dehydrogenase mutant gene, which is characterized in that: the nucleotide sequence of the 4-xylitol dehydrogenase mutant gene is shown as SEQ ID NO: 4, respectively.

3. A recombinant vector comprising the 4-xylitol dehydrogenase mutant gene according to claim 2.

4. A genetically engineered bacterium producing the 4-xylitol dehydrogenase mutant according to claim 1, characterized in that: the genetically engineered bacterium comprises the 4-xylitol dehydrogenase mutant gene of claim 2 or the recombinant vector of claim 3.

5. The genetically engineered bacterium of claim 4, wherein: the host cell of the genetic engineering bacteria is Escherichia coli BL21(DE3) cell.

6. Use of the 4-xylitol dehydrogenase mutant gene of claim 2, the recombinant vector of claim 3 or the genetically engineered bacterium of any one of claims 4 to 5 in the preparation of 4-xylitol dehydrogenase.

7. A preparation method of a 4-xylitol dehydrogenase mutant is characterized by comprising the following steps: the method comprises the following steps: culturing the genetically engineered bacterium of claim 4 or 5 to obtain a recombinant expressed 4-xylitol dehydrogenase mutant.

8. Use of the 4-xylitol dehydrogenase mutant according to claim 1 for the production of L-xylulose by the conversion of xylitol.